Circulating CD36+ microparticles are not altered by docosahexaenoic or eicosapentaenoic acid supplementation

Nutrition, Metabolism & Cardiovascular Diseases (2016) 26, 254e260

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Circulating CD36þ microparticles are not altered by docosahexaenoic or eicosapentaenoic acid supplementation* M. Phang a, R.F. Thorne b,c, M.J. Alkhatatbeh d, M.L. Garg a,c, L.F. Lincz c,e,* a

Nutraceuticals Research Group, School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW 2308, Australia School of Environmental and Life Sciences, University of Newcastle, Ourimbah, NSW 2258, Australia c Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia d Clinical Pharmacy Department, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid, Jordan e Hunter Haematology Research Group, Calvary Mater Newcastle Hospital, Waratah, NSW 2298, Australia b

Received 21 July 2015; received in revised form 20 November 2015; accepted 10 December 2015 Available online 18 December 2015

KEYWORDS Microparticles; Scavenger receptor; CD36; Polyunsaturated fatty acids; Docosahexaenoic acid; Eicosapentaenoic acid

Abstract Background and aims: Circulating microparticles (MP) are the source of a plasma derived form of the scavenger receptor CD36, termed soluble (s)CD36, the levels of which correlate with markers of atherosclerosis and risk of cardiovascular disease. Long chain n-3 polyunsaturated fatty acids have cardioprotective effects that we have previously reported to be gender specific. The aim of this study was to determine if dietary docosahexaenoic acid (DHA) and/or eicosapentaenoic acid (EPA) supplementation affect circulating CD36 þ MP levels, and if this occurs differentially in healthy men and women. Methods and results: Participants (43M, 51F) aged 39.6  1.7 years received 4 weeks of daily supplementation with DHA rich (200 mg EPA; 1000 mg DHA), EPA rich (1000 mg EPA; 200 mg DHA), or placebo (sunola) oil in a double-blinded, randomised, placebo controlled trial. Plasma CD36 þ MP were enumerated by flow cytometry and differences between genders and treatments were evaluated by Student’s or paired t-test and one way ANOVA. Males and females had similar levels of CD36 þ MP at baseline (mean Z 1018  325 vs 980  318; p Z 0.577) and these were not significantly changed after DHA (M, p Z 0.571; F, p Z 0.444) or EPA (M, p Z 0.361; F, p Z 0.901) supplementation. Likewise, the overall percent change in these levels were not different between supplemented cohorts compared to placebo when all participants were combined (% change in CD36 þ MP: DHA Z 5.7  37.5, EPA Z 3.4  35.4, placebo Z 11.5  32.9; p Z 0.158) or stratified by gender (M, DHA Z 2.6  30.6, EPA Z 15.1  20.1, placebo Z 21.4  28.7, p Z 0.187; F, DHA Z 11.7  41.5, EPA Z 6.8  42.9, placebo Z 2.8  34.7, p Z 0.552). Conclusion: The cardioprotective effects of DHA and EPA do not act through a CD36 þ MP mechanism. ª 2015 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.

Abbreviations: MP, microparticles; PA, palmitic Acid; SA, stearic acid; OA, oleic acid; LA, linoleic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. *

Australian New Zealand Clinical Trials Registry: ACTRN12611000098932 (28/01/2011). * Corresponding author. Hunter Haematology Research Group, Calvary Mater Newcastle Hospital, Waratah, NSW 2298, Australia. Tel.: þ61 2 40143049; fax: þ61 2 4960 2136. E-mail address: [email protected] (L.F. Lincz). http://dx.doi.org/10.1016/j.numecd.2015.12.003 0939-4753/ª 2015 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.

DHA, EPA and circulating CD36

Introduction Inflammation is thought to play a central role in the development of atherosclerosis and coronary artery disease [1]. Plaque formation is promoted when reactive oxygen and nitrogen species within the vessel lead to the generation of oxidised LDL which is internalised by macrophages and leads to the formation of lipid laden foam cells which eventually form part of the atherosclerotic plaque. A key role in this process is performed by the multifunctional transmembrane signalling molecule, CD36 (also known as Fatty Acid Translocase), that acts as a scavenger receptor for both native and atherogenic lipoproteins including oxidised LDL and HDL [reviewed in Ref. [2]]. Expression of CD36 on macrophages becomes upregulated in the presence of oxLDL, further exacerbating the atherosclerotic process. Although normally found expressed on the surface of cells and tissues such as platelets, erythrocytes, monocytes, endothelial cells and leukocytes [1], a circulating form of CD36, termed soluble (s) CD36, has been identified in human plasma [3]. Plasma sCD36 is believed to reflect tissue levels of the receptor and was found to be correlated with atherosclerotic plaque instability, a major determinant in the risk of acute coronary syndromes [4]. Even in a healthy population, sCD36 has been significantly associated with markers of insulin resistance and atherosclerosis [5]. The heavily N-glycosylated extracellular domain of CD36 renders the glycoprotein highly resistant to proteolytic cleavage [6]. Using immunological methods we previously established that all detectable sCD36 was intact and entirely associated with circulating microparticles (MP) [7]. These small (0.1e1 mm diameter) membrane bound vesicles are released from activated and/or apoptotic cells, and often express phosphatidylserine as well as many of the antigens from their original cell of origin [8]. In normal healthy individuals the majority of circulating MP are platelet derived [9], however, their numbers and cellular source change dramatically in pathological states, especially those characterised by inflammation, vascular dysfunction and thrombosis; including the metabolic syndrome, diabetes and cardiovascular disease [10]. The ability of such microparticles to adversely affect other cells in the body has been clearly illustrated by in vitro experiments showing the inhibition of endothelial tube formation in the presence of MP from diabetic patients with coronary artery disease [11] and in vivo induction of endothelial dysfunction in mice when injected with MP from humans with metabolic syndrome [12]. In regards to CD36 we have shown that enumeration of the number of circulating CD36 þ MP is more closely related to diabetic status than levels of sCD36 protein per se [13]. Thus CD36 may identify a specific subset of inflammatory associated MP that act as mediators in the pathogenesis of cardiovascular disease. The long chain n-3 polyunsaturated fatty acids (n-3 PUFAs), docosahexaenoic acid, C22:6n-3 (DHA) and eicosapentaenoic acid, C20:5n-3 (EPA) have anti-atherogenic,

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anti-inflammatory and anti-thrombotic properties that are believed to contribute to prevention of cardiovascular disease [14]. These cardioprotective effects have recently been shown to be independent of sCD36 protein levels in a group of overweight subjects participating in a 6 week placebo controlled trial of combination marine n-3 PUFA supplementation [15]. In addition to the increased accuracy afforded through measurement of CD36 þ MP over sCD36 protein, our own studies have indicated that some of the beneficial effects of the DHA and EPA contained in n-3 PUFA formulations are both differential and gender specific [16]. Taken together, we hypothesised that any specific effect of DHA or EPA in these previous cohorts may have been overlooked. Thus the aim of this study was to determine if DHA and/or EPA alone affect circulating CD36 þ MP levels, and if this occurred differentially in healthy men and women. Methods Study population This study was a double blinded, placebo controlled intervention trial, details of which have been previously reported [16]. A total of 94 healthy participants completed the study; 43 males and 51 females, recruited from the general community of Newcastle, NSW, Australia. Exclusion criteria were: diagnosed non-insulin dependent diabetes; insulin resistance; impaired glucose tolerance; cardiovascular or haematological disorders; body mass index (BMI) greater than 35 kg/m2; taking aspirin, anti-platelet medication or non-steroidal anti-inflammatory drugs. Participants were also excluded if they had consumed fish oil supplements or consumed more than two seafood servings per week. Participants were asked to complete a medical questionnaire, 24 h food recall and follow a diet low in tomatoes and seafood as well as maintain their usual physical activity during the intervention and especially in the 24 h prior to the study days. All participants provided written informed consent according to governmental regulations concerning the ethical use of human volunteers. Approval for the study was granted by the Human Research Ethics Committee of the University of Newcastle, Australia and it was registered in the Australian New Zealand Clinical Trials Registry (ACTRN12611000098932) on 28/01/2011. The study was conducted in accordance with The Declaration of Helsinki. Participants were randomised with gender stratification to a treatment protocol of 2  1 g capsules daily for 4 weeks of supplements containing either: Placebo (Sunola oil); DHA rich oil [100:500 mg EPA/DHA (EPAX 1050 TG/N)] or EPA rich oil [500:100 mg EPA/DHA (EPAX 5510 TG/N)]. Blood analysis Venous blood was collected into vacutainer tubes following a >10 h fast. Whole blood in 3.2% sodium citrate

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was collected for microparticle enumeration, centrifuged at 3000  g for 10 min to obtain platelet free plasma and aliquots were stored at 80  C until further analysis. For quantification of plasma fatty acid composition, blood was collected into EDTA tubes and prepared as above. Fasting blood was collected into EDTA and lithium heparin tubes for analysis of full blood count and hormonal levels (testosterone, oestradiol), respectively. Samples were analysed by Hunter New England Area Health Pathology Services (NSW, Australia) using standard analytical techniques. Plasma fatty acid analyses The fatty acid composition of plasma lipids was determined using an acetyl chloride methylation procedure as previously described [16]. Fatty acid methyl esters were quantified using gas chromatography (Hewlett Packard 6890; Hewlett Packard, Palo Alto, CA, USA). The identity of each fatty acid peak was ascertained by comparison of the peaks’ retention time with those of synthetic standards of known fatty acid composition (Nu Check Prep, Elysian, MN, USA). The relative amount of each fatty acid was quantified by integrating the area under the peak and dividing the result by the total area for all fatty acids. Fatty acid results are reported as percentage of total fatty acids.

and 3 mm diameter fluorescent beads (Megamix, Biocytex, Marseille, France) was used to ensure adequate FSC resolution and set the MP detection limits to exclude events >0.9 mm in diameter according to the manufacturer’s instructions. A 10 ml aliquot of platelet free plasma was incubated at room temperature for 30 min with an antibody against CD36 (clone 11H5 [18]) conjugated to DyLight-488 in combination with anti CD41-PE (clone PL2-49, Biocytex; platelet marker) and Annexin V-APC (eBioscience, San Diego, CA, USA; to measure phosphatidylserine exposure). All assays were diluted in calcium rich binding buffer as supplied by the manufacturer (eBioscience). A known number of CountBright fluorospheres (Molecular Probes, Eugene, Oregon, USA), were used to enable absolute MP quantification and events were collected for 60 s at low flow rate on a BD FACS Canto (BD Biosciences) prior to analysis using FACS Diva software (BD Biosciences). The total numbers of CD36þ events were quantified and the absolute number of MP in each plasma sample was calculated using the formula: MP/ml Z (MP count/bead count)  (total beads/test volume). The reproducibility of the assay was determined by staining and analysing a series of 5 different plasma samples in triplicate on five separate occasions. The intra- and interassay variability was thus calculated to be 11.3% and 10.6%, respectively.

Analysis of MP by flow cytometry

Statistical analysis

Staining and analysis of MP was performed essentially as previously described [13] and according to guidelines established by the International Society on Thrombosis and Haemostasis Vascular Biology SSC on the Standardization of FMC-based PMP enumeration by flow cytometry [17]. To calibrate the cytometer, a blend of 2:1:1 0.5, 0.9

All data are presented as mean  standard deviation (SD). Preliminary assumption testing was conducted to check for normality, linearity, outliers and homogeneity of variance with no serious violations observed for all test variables within gender and supplement groups. Differences between genders/treatments were evaluated by Student’s

Table 1 Baseline characteristics and fatty acid profiles (expressed as % of total fatty acid). Males þ females (n Z 94) Mean  SD

Males (n Z 43) Mean  SD

Age (years) BMI (kg/m2)

39.6  16.9 24.6  3.7

C16:0 PA C18:0 SA C18:1n-9 OA C18:2n-6 LA C20:4n-6 AA 20:5n-3 EPA 22:6n-3 DHA

20.5 7.4 20.9 27.7 7.4 1.2 2.7

      

2.2 1.0 2.6 3.6 1.4 0.7 1.1

20.4 7.3 21.3 27.2 7.4 1.3 2.8

      

2.0 0.8 3.0 3.9 1.4 0.8 1.1

SFA MUFA n-6 PUFA n-3 PUFA Ratio n-6:n-3

28.2 25.1 40.0 6.1 6.9

    

2.3 3.2 4.2 1.7 2.0

28.0 25.1 39.6 6.5 6.5

    

2.1 3.6 4.5 1.8 1.9

CD36 þ MP *p < 0.05.

997.3  320.0

40.8  18.1 25.6  3.8

1017.6  324.6

Females (n Z 51) Mean  SD

p-Value (M vs F)

38.6  15.9 23.7  3.5

0.523 0.013*

20.5 7.5 20.6 28.1 7.4 1.0 2.6

      

2.3 1.1 2.2 3.3 1.4 0.5 1.1

0.887 0.248 0.185 0.218 0.971 0.048* 0.520

28.3 25.1 40.3 5.9 7.4

    

2.5 2.9 4.0 1.5 2.0

0.561 0.929 0.445 0.079 0.033*

979.8  318.3

0.577

DHA, EPA and circulating CD36

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Table 2 Percent change in CD36 þ MP and plasma fatty acids after 4 weeks supplementation. Males þ females

DHA (n Z 31) Mean  SD

EPA (n Z 31) Mean  SD

C16:0 PA C18:0 SA C18:1n-9 OA C18:2n-6 LA C20:4n-6 AA 20:5n-3 EPA 22:6n-3 DHA

3.8 4.3 8.0 0.2 3.8 183.1 161.6

      

8.1 9.6 8.4* 8.9 13.2 165.1* 246.3*y

0.5 2.4 2.7 4.2 4.0 230.2 32.3

      

7.1 12.5 9.6y 9.7y 12.8 157.4y 41.8*

0.9 1.8 5.6 2.1 1.0 3.2 3.5

      

8.3 14.0 12.4*y 9.7y 12.1 30.0*y 31.3y

0.058 0.136 0.000 0.031 0.573 0.000 0.000

SFA MUFA n-6 PUFA n-3 PUFA n-6:n-3

1.0 8.3 1.2 70.5 32.4

    

9.3 8.0* 7.0 56.1* 41.1*

0.0 3.6 4.8 55.9 31.8

    

7.2 9.5y 7.7y 38.2y 33.1y

0.4 2.9 1.0 4.8 10.5

    

7.5 11.1*y 8.9y 18.7*y 25.9*y

0.902 0.000 0.016 0.000 0.000

CD36 þ MP

5.7  37.5

3.4  35.4

Placebo (n Z 32) Mean  SD

11.5  32.9

p-Value (ANOVA)

0.158

Values within a row sharing a common symbol are significantly different p < 0.05 (post-hoc Tukey test). Individual absolute p-values are provided in the text. *p < 0.05.

t-test or one-way analysis of variance (ANOVA) with post-hoc Tukey HSD (honest significant difference) test. Bivariate correlations were determined using Pearson Product-Moment coefficients. All calculations were performed with Statistica v10.0 (StatSoft, Tulsa, OK, USA) using two-tailed tests, and p-values < 0.05 were considered statistically significant.

Results Baseline demographics Table 1 lists the baseline demographics, fatty acid profiles and circulating CD36 þ MP for the entire cohort with and without stratification by gender. The average participant age was 39.6  16.9 years, with a BMI of 24.6  3.7 kg/m2. Males and females were well matched for age, but males had significantly greater BMI (25.6  3.8 kg/m2) than females (23.7  3.5 kg/m2,; p Z 0.013). Baseline plasma fatty acid levels were also similar between the genders, with the exception of slightly higher levels of EPA (1.3  0.8 vs 1.0  0.5; p Z 0.048), and a lower ratio of n-6:n-3 PUFA in males (6.5  1.9 vs 7.4  2.0; p Z 0.033). Mean CD36 þ MP levels were calculated to be 997.3  320.0/ul of plasma, and these did not differ significantly between males and females (p Z 0.577). When assigned to each treatment group, there were no significant differences in these measures between the males in each treatment group or between the females in each group, and no baseline differences were observed with subgroup analyses for gender within and between treatment groups for DHA, n Z 31 (M:F Z 15:16); EPA, n Z 31 (M:F Z 13:18); Placebo, n Z 32 (M:F Z 15:17) (data not shown). There were only 3 habitual smokers in the cohort: 1 female and 2 males, stratified to EPA, DHA and placebo treatment groups, respectively. This was not

considered enough to be a confounding factor and was omitted from subsequent analysis.

Post supplementation After 4 weeks supplementation with either DHA or EPA, there was a significant decrease in C18:1n-9 (oleic acid; 8.0  8.4%, p < 0.001 and 2.7  9.6%, p Z 0.005, respectively), monounsaturated fatty acids (8.3  8.0%, p < 0.001 and 3.6  9.5%, p Z 0.024) and n-6:n-3 ratio (32.4  41.1%, p < 0.001 and 31.8  33.1%; p < 0.001), with a concomitant increase in EPA (183.1  165.1%, p < 0.001 and 230.2  157.4%, p < 0.001) and n-3PUFA (70.5  56.1%, p < 0.001 and 55.9  38.2%, p < 0.001) compared to placebo (Table 2). Participants taking DHA had a much larger change in plasma DHA levels (161.6  246.3%) compared to those on EPA (32.3  41.8%, p Z 0.002) or placebo (3.2  30.0%, p < 0.001), while only the EPA cohort experienced a significant decrease in C18:2n-6 (linoleic acid; 4.2  9.7%) and n-6 PUFA (4.8  7.7%) compared to those on placebo (LA Z 2.1  9.7%, p Z 0.027 and n-6 PUFA Z 1.0  8.9%, p Z 0.012). The overall percent change in CD36 þ MP levels were not different between supplement cohorts compared to placebo when all participants were combined (M þ F: DHA Z 5.7  37.5%, EPA Z 3.4  35.4%, placebo Z 11.5  32.9%, p Z 0.158; Table 2) or stratified by gender (M: DHA Z 2.6  30.6%, EPA Z 15.1  20.1%, placebo Z 21.4  28.8%, p Z 0.187 and F: DHA Z 11.8  41.5%, EPA Z 6.8  42.9%, placebo Z 2.8  34.7%, p Z 0.552; Fig. 1). In addition to total CD36 þ MP, we were able to also determine the absolute number of each individual MP species analysed (total CD41þ, total Annexin V, CD41þ/Annexin Vþ, CD36þ/CD41þ and CD36þ/Annexin Vþ) and found no

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Figure 1 EPA and/or DHA supplementation did not significantly affect % change in CD36 þ MP overall or when stratified by gender. Box plots indicate mean  SD with bars showing 1.96*SD of percent change in CD36 þ MP levels after 4 weeks supplementation as indicated. p Z 0.158, 0.187 and 0.552 for males þ females, males, and females, respectively after treatment with EPA or DHA compared to placebo.

difference in any of these MP subsets between males and females at baseline or after 4 weeks supplementation (data not shown). Although there was a significant correlation between CD36 þ MP and C18:0 plasma levels at baseline (R Z 0.27, p Z 0.009), there was no correlation between the change in CD36 þ MP and the change in this or any other individual fatty acid level after 4 weeks supplementation (Table 3).

Discussion Although dietary fat is essential for good health, the type of fat consumed as reflected by plasma fatty acid profiles has been associated with many of the risk factors of cardiovascular disease, including obesity, insulin resistance and the metabolic syndrome [19]. The cardioprotective

Table 3 Correlation between CD36 þ MP and plasma fatty acid profiles at baseline and after 4 weeks supplementation. Variable

Baseline variable correlated with baseline CD36 þ M %

% Change in variable correlated with % change CD36 þ MP

R

p-Value

R

p-Value

Age BMI

0.14 0.09

0.192 0.386

C16:0 PA C18:0 SA C18:1n-9 OA C18:2n-6 LA C20:4n-6 AA 20:5n-3 EPA 22:6n-3 DHA

0.20 0.27 0.05 0.16 0.06 0.05 0.04

0.062 0.009* 0.633 0.130 0.563 0.658 0.685

0.01 0.19 0.06 0.04 0.15 0.07 0.12

0.917 0.068 0.545 0.708 0.153 0.525 0.243

SFA MUFA n-6 PUFA n-3 PUFA n-6:n-3

0.06 0.02 0.05 0.06 0.00

0.541 0.855 0.616 0.562 0.967

0.09 0.08 0.01 0.10 0.01

0.367 0.470 0.907 0.356 0.932

effects of n-3 PUFA are believed to be multifactorial, attributable to their anti-inflammatory properties and direct effects on blood lipids, cardiomyocytes and vascular epithelium [20]. Biologically, ingested EPA and DHA are quickly incorporated into the phospholipid membranes of cells involved in inflammation where biochemically they compete with the more abundant n-6 PUFA, AA, as a substrate for cyclooxygenase-2, resulting in decreased production of pro-inflammatory mediators [21]. This has led to the proposed use of the ratio of n-6:n-3 as a more useful measure of PUFA status [22]. Whether these biochemical processes can be directly translated into improved health outcomes remains controversial, as two recent meta analyses have found insufficient evidence for n-3 PUFA supplementation to prevent cardiovascular events [23,24]. However the majority of human trials have employed fish oil supplements containing mixtures of differing ratios of DHA:EPA, which we have previously shown to have differential sex-specific responses that could account for such discrepancies [16]. Circulating MP levels have been shown to increase postprandially in humans in response to various foods, especially fats [25]. In addition, these changes may be influenced by gender. For instance, we have previously shown that platelet MP procoagulant activity can be quickly reduced in healthy men by ingestion of fish oil. In this setting, a single 2 g dose of EPA (but not DHA) rich oil significantly decreased MP activity in males, whereas neither was effective in females [26]. The effects of longer term supplementation with fish oil has more recently been shown to decrease circulating levels of endothelial derived MP in participants with moderate risk of cardiovascular disease, indicating a reduction in endothelial damage [27]. In addition, treatment with n-3 PUFA after myocardial infarct reduced levels of platelet- and monocyte-derived microparticles, further supporting their anti-inflammatory and anti-thrombotic properties [28]. In the present study we found no significant change in any platelet derived subsets of MP after 4 weeks supplementation with EPA or DHA compared to placebo. It is presumable that our cohort of healthy individuals

DHA, EPA and circulating CD36

would have normal levels of MP prior to supplementation so that further reduction may not occur. Despite this possibility, we have previously shown that this is not the case for other haemostatic markers of thrombotic risk. In particular, this same cohort of healthy individuals exhibited a gender specific differential effect of n-3 PUFA supplementation on platelet aggregation whereby aggregation was reduced after supplementation with EPA but not DHA in men, and with DHA but not EPA in women [16]. Our results are consistent with those of Veno et al. who found no change in circulating CD36 protein levels after 6 weeks of n-3 PUFA supplementation in overweight individuals [15]. Since the majority of detectable circulating CD36 þ MP in our assays co-expressed the platelet CD41a marker, our results also reflect those of Serebruany et al., whereby 2 week supplementation with n-3 PUFA in patients with coronary artery disease did not alter CD36 expression on platelets [29]. In contrast, incorporation of EPA and DHA into the cell membranes of human monocytes has been shown to significantly reduce their expression of CD36 [30], suggesting that specific investigation of circulating monocyte derived CD36 þ MP may be more informative for future studies. In addition, others have shown that the many known haplotypes of CD36 can influence individual responses to the beneficial effects of fish oil supplementation on plasma triacyl glycerol and HDL cholesterol [31]. Thus it is possible that CD36 genotypes, which remained unidentified in the present study, also influence circulating levels and/or MP expression and these should be considered in any future study designs. There are several limitations to this study. Firstly, although flow cytometry is the most widely adopted method for identifying MP, it is limited in its ability to reliable detect such small particles. Thus CD36 þ MP populations below approximately 500 nm in diameter are not considered in this survey and may well be the major subsets affected by DHA and/or EPA supplementation. In addition, the use of polystyrene beads to set size gates introduce another level of error as they are known to have a higher reflective index compared to that of biological particles [32]. However, in the absence of biological standards, these remain the most appropriate tool available, and although more sensitive and specialised technologies are emerging, these currently lack the ability to identify MP subsets via their multiple surface antigens. Finally, although we were careful to exclude participants with personal evidence or clinical risk factors for CVD, we did not inquire about their family history. Others have shown that healthy first degree relatives of patients with premature CVD have elevated levels of endothelial MP [33], suggesting that a predisposition to CVD could potentially impact on MP dynamics. In conclusion, our results do not support a CD36 þ MP mediated mechanism for the cardioprotective effects of DHA and EPA, but cannot rule out the possible involvement of other MP subsets, which would be worth investigating in future studies.

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Acknowledgements This work was funded in part by a Lions District 201N3 Diabetes Foundation grant (HMRI 10-08) administered through the Hunter Medical Research Institute. References [1] Silverstein RL. Inflammation, atherosclerosis, and arterial thrombosis: role of the scavenger receptor CD36. Cleve Clin J Med 2009; 76(Suppl. 2):S27e30. http://dx.doi.org/10.3949/ccjm.76.s2.06. [2] Thorne RF, Mhaidat NM, Ralston KJ, Burns GF. CD36 is a receptor for oxidized high density lipoprotein: implications for the development of atherosclerosis. FEBS Lett 2007;581:1227e32. S00145793(07)00209-8 [pii], http://dx.doi.org/10.1016/j.febslet.2007.02. 043. [3] Handberg A, Levin K, Hojlund K, Beck-Nielsen H. Identification of the oxidized low-density lipoprotein scavenger receptor CD36 in plasma: a novel marker of insulin resistance. Circulation 2006;114: 1169e76. http://dx.doi.org/10.1161/CIRCULATIONAHA.106.626135. [4] Handberg A, Skjelland M, Michelsen AE, Sagen EL, KrohgSorensen K, Russell D, et al. Soluble CD36 in plasma is increased in patients with symptomatic atherosclerotic carotid plaques and is related to plaque instability. Stroke 2008;39:3092e5. http://dx. doi.org/10.1161/STROKEAHA.108.517128. [5] Handberg A, Hojlund K, Gastaldelli A, Flyvbjerg A, Dekker JM, Petrie J, et al. Plasma sCD36 is associated with markers of atherosclerosis, insulin resistance and fatty liver in a nondiabetic healthy population. J Intern Med 2011;217(3):294e304. http://dx. doi.org/10.1111/j.1365-2796.2011.02442.x. [6] Gruarin P, Thorne RF, Dorahy DJ, Burns GF, Sitia R, Alessio M. CD36 is a ditopic glycoprotein with the N-terminal domain implicated in intracellular transport. Biochem Biophys Res Commun 2000;275: 446e54. S0006-291X(00)93333-3 [pii], http://dx.doi.org/10.1006/ bbrc.2000.3333. [7] Alkhatatbeh MJ, Mhaidat NM, Enjeti AK, Lincz LF, Thorne RF. The putative diabetic plasma marker, soluble CD36, is non-cleaved, non-soluble and entirely associated with microparticles. J Thromb Haemost 2011;9(4):844e51. http://dx.doi.org/10.1111/j. 1538-7836.2011.04220.x. [8] Burnier L, Fontana P, Kwak BR, Angelillo-Scherrer A. Cell-derived microparticles in haemostasis and vascular medicine. Thromb Haemost 2009;101:439e51. http://dx.doi.org/09030439. [9] Horstman LL, Ahn YS. Platelet microparticles: a wide-angle perspective. Crit Rev Oncol Hematol 1999;30:111e42. S10408428(98)00044-4 [pii]. [10] Nomura S, Shimizu M. Clinical significance of procoagulant microparticles. J Intensive Care 2015;2:3. http://dx.doi.org/10. 1186/s40560-014-0066-z. [11] Tsimerman G, Roguin A, Bachar A, Melamed E, Brenner B, Aharon A. Involvement of microparticles in diabetic vascular complications. Thromb Haemost 2011;106:310e21. http://dx.doi. org/10.1160/th10-11-0712. [12] Agouni A, Lagrue-Lak-Hal AH, Ducluzeau PH, Mostefai HA, Draunet-Busson C, Leftheriotis G, et al. Endothelial dysfunction caused by circulating microparticles from patients with metabolic syndrome. Am J Pathol 2008;173:1210e9. http://dx.doi.org/10.2353/ ajpath.2008.080228. [13] Alkhatatbeh MJ, Enjeti AK, Acharya S, Thorne RF, Lincz LF. The origin of circulating CD36 in type 2 diabetes. Nutr Diabetes 2013; 3:e59. http://dx.doi.org/10.1038/nutd.2013.1. [14] Lorente-Cebrián S, Costa AV, Navas-Carretero S, Zabala M, Martínez JA, Moreno-Aliaga M. Role of omega-3 fatty acids in obesity, metabolic syndrome, and cardiovascular diseases: a review of the evidence. J Physiol Biochem 2013;69:633e51. http:// dx.doi.org/10.1007/s13105-013-0265-4. [15] Veno SK, Nielsen MR, Lundbye-Christensen S, Schmidt EB, Handberg A. The effect of low-dose marine n-3 fatty acids on plasma levels of sCD36 in overweight subjects: a randomized, double-blind, placebo-controlled trial. Mar Drugs 2013;11: 3324e34. http://dx.doi.org/10.3390/md11093324. [16] Phang M, Lincz LF, Garg ML. Eicosapentaenoic and docosahexaenoic acid supplementations reduce platelet aggregation and

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[17]

[18]

[19]

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